Nuclear Medicine and Biology 31 (2004) 477– 482

www.elsevier.com/locate/nucmedbio

Transcellular transport of 4-iodo-L-meta-tyrosine via system L acrossmonolayers of kidney epithelial cell Line LLC-PK1
Naoto Shikanoa,*, Keiichi Kawaib, Syuichi Nakajimaa, Akiko Kuboderac, Nobuo Kubotaa,
Nobuyoshi Ishikawaa, Hideo Sajida

Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japanb
School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japanc
Faculty of Pharmaceutical Sciences, Science University of Tokyo, 12 Ichigaya Funagawara-machi, Shinjuku-ku, Tokyo 162-0826, Japand
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8151, Japan
Received 8 October 2003; received in revised form 11 December 2003; accepted 19 December 2003

Abstract
The substance 4-[125I]iodo-L-meta-tyrosin (4-[125I]mTyr) is a radioiodinated amino acid that exhibits high in vivo stability and rapid
renal elimination in vivo. We investigated transport of 4-[125I]mTyr in LLC-PK1 (porcine kidney epithelial cell line) monolayers grown on
collagen-coated, micro-porous membrane filters. We found that 4-[125I]mTyr transport in LLC-PK1 cells was carrier-mediated andsodium-independent, and that 4-[125I]mTyr transport was similar to that of L-Tyr and 3-iodo-␣-methyl-L-tyrosine. The results of theinhibition experiments suggest that 4-[125I]mTyr transport is predominantly mediated by a L-type amino acid transporter 1–like porcine
homologue (a component of system L) in both basolateral and apical membrane. © 2004 Elsevier Inc. All rights reserved.
Keywords: Amino acid transport; System L; Epithelial cell line; LLC-PK1; 4-Iodo-L-meta-tyrosine

1. Introduction
In a recent study, we found that 4-[125I]iodo-L-metatyrosine (4-[125I]mTyr; Fig. 1), an artificial amino acid,exhibits high metabolic stability and similar biodistribution
to other radiolabeled L-Tyr analogs [1]. Recent reports
indicate that 4-[125I]mTyr shows promise as a new aminoacid transport marker.
Christensen [2] reported that three different transportsystems (designated A, ASC and L) mediate transport ofneutral amino acids across the plasma membrane of mammalian cells. System A is Na⫹-dependent and most
readily transports neutral amino acids with short, polar,
or linear side chains. Specific inhibitors of system A are
2-(methylamino)isobutyric acid (MeAIB) and 2-aminoisobutyric acid (AIB). System ASC is also Na⫹-dependent but excludes MeAIB. System L is Na⫹-independent,
and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid
(BCH) acts as a specific substrate. System L is essential* Corresponding author. Tel.: ⫹81-29-840-2217; fax: ⫹81-29-8402317.
E-mail address: sikano@ipu.ac.jp (N. Shikano).
0969-8051/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2003.12.006

for cellular nutrition and trans-cellular transport of neutral amino acids at blood-tissue barriers of epithelia.
LLC-PK1, an epithelial cell line from a Hampshire pig
kidney, has a hexose and phosphate transport system withcharacteristics similar to those observed in the proximal
tubule [3-6]. Confluent monolayers of LLC-PK1 cells retain
several of the characteristics of differentiated renal proximal
tubular cells. For example, LLC-PK1 forms an orientedmonolayer with tight junctions and absorbs electrolytes andsome nutrients via the microvilli of the apical membrane[6]. LLC-PK1 cells are polarized epithelial cells with apicaland basolateral membrane domains [3-6]. Incidentally, several characteristics of LLC-PK1 cells are comparable tothose of tumor cell lines. It has been reported that LLC-PK1
cells grow rapidly in monolayer cultures. They are stablethrough more than 300 serial passages, and there is noevidence that the cells undergo transformation [6]. We speculated that studies of transcellular transport of artificial
amino acids could be an informative new application of the
LLC-PK1 system. The purpose of the present study was toclarify mechanisms of transcellular transport of 4-[125I]mTyr by LLC-PK1 cell monolayers grown on collagencoated, microporous membrane filters [3] (Fig. 2).

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N. Shikano et al. / Nuclear Medicine and Biology 31 (2004) 477– 482

Fig. 1. Chemical structure of 4-[125I]iodo-L-meta-tyrosine.

2. Methods and materials
2.1. Materials
D,L-mTyr, BCH, MeAIB, AIB, and chloramine-T wereacquired from Sigma-Aldrich Japan K.K. (Tokyo, Japan).
Natural L-amino acids and other chemicals of reagent gradewere purchased from Kanto Chemical Co. (Tokyo, Japan).
[125I]NaI was obtained from Amersham Pharmacia Biotech
UK (Buckinghamshire, UK). A Crownpak CR(⫺), a chiralcolumn, (4 ⫻ 150 mm; Daicel Chemical Industries, Tokyo,
Japan) and a Nova-Pak C18 (3.9 ⫻ 300 mm; Waters, Milford, MA) were used for separation and purification. A
silica gel, thin-layer chromatography kit (TLC, catalogue
number Art. 5553) was obtained from Merck (Darmstadt,
Germany).
For cell line studies, LLC-PK1 cells were obtained from
Dainippon Pharmaceuticutical Co. (Osaka, Japan). Plastic
tissue culture dishes (diameter 60 mm; Falcon; Becton
Dickinson, Lincoln Park, NJ), plastic culture flasks (surface
area, 25 cm2; Nalge Nunc International, Roskilde, Denmark) and Transwell cell chambers (surface area, 4.71 cm2;
Costar, Cambridge, MA) were purchased.
2.2. Preparation of labeled compounds
Preparation of 4-[125I]mTyr was performed using a previously reported method [7,8]. Briefly, the racemic body ofmTyr was separated by HPLC using Crownpak CR (⫺).
Chloramine-T (10 ␮L at 1.0 mmol/L in 0.05 mol/L phosphate buffer [pH 8.5]) was added to a mixture of L-mTyr(100 ␮L at 1.0 mmol/L) and was carrier free [125I]NaI(3.7-7.4 MBq) in 35 ␮L of 0.4 mol/L phosphate buffer(pH 8.5). The resulting solution was allowed to stand for
30 minutes, and 10 ␮L of 1.0 mmol/L sodium metabisulfite in 0.05 mol/L phosphate buffer (pH 8.5) was added.
Geometric isomer separation by HPLC was then performed using a Nova-Pak C18 (eluent, 0.02 mol/L potassium acetate: ethanol [90:1]; flow rate, 0.5 mL/min; retention time for 4-isomer, 17-20 min; retention time forI-, 4-5 min; retention time for cold mTyr, 5-7 min) [8].
Labeling efficiency and radiochemical purity were studied using silica gel TLC with two solvent systems: 1)
methanol:acetic acid (100:1) (Rf value: 4-[125I]mTyr,0.50; I-, 0.80); and 2) methanol:10% ammonium acetate(10:1) (Rf value: 4-[125I]mTyr, 0.55; I-, 0.80).
2.3. Cell cultures
Cell line studies were performed using modified methods
described by Saitoh et al. [9], as follows. LLC-PK1 cells
were maintained by serial passages in 25-cm2 cell culture
flasks. The cells were fed with Dulbecco’s modified Eagle’s
medium (Sigma-Aldrich Japan K.K., Tokyo, Japan) supplemented with L-glutamine (2 mmol/L) and 10% fetal bovine
serum without antibiotics, in an atmosphere of 7.5% CO2
and 95% air at 37°C (pH 7.4). Subculturing was performed
every 5 days using 0.02% EDTA and 0.05% trypsin. Cells
were used between passages 230 and 240.
2.4. Monolayer preparation
To prepare monolayers on a micropores support, cells
were seeded at a density of 5 ⫻ 105 cells/cm2 on polycarbonate membrane filter (pores, 3 ␮m) in Transwell cellchambers. The volume of medium inside and outside theTranswell chamber was 1.5 and 2.6 mL, respectively. Freshmedium was replaced every 2 days. The cells were used
between days 4 and 5.
2.5. Measurement of transepithelial transport and cellularaccumulation

Fig. 2. LLC-PK1 cell monolayer grown on a permeable support in Transwell cell chambers. It provides a cell culture model that exhibits several
characteristics of in vivo epithelial membranes.

Transepithelial transport and accumulation of 4-[125I]mTyr were measured using monolayer cultures grown in theTranswell chambers. The incubation medium was Dulbecco’s phosphate-buffered saline (PBS; pH 7.4) containing
137 mmol/L NaCl, 3 mmol/L KCl, 8 mmol/L Na2HPO2, 1
mmol/L CaCl2, and 0.5 mmol/L MgCl. The pH of medium
was adjusted by adding HCl or NaOH solution. In Na⫹-free
medium, the NaCl and Na2HPO4 normally present in PBS
were replaced with choline chloride and K2HPO4, respectively. In general experiments, after the removal of culture
medium from both sides of the monolayers, the cell mono-

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layers were preincubated with 2 mL of incubation medium
on each side for 10 minutes at 37°C. Then, 2 mL of incubation medium containing 4-[125I]mTyr (18.5 kBq) was
added either to the basolateral or apical side, and 2 mL ofradioactive free incubation medium were added to the opposite side. The monolayers were incubated for a specified
period of time at 37°C or 4°C. [14C]Inulin (18.5 kBq), a
compound that is not transported by the cells, was used toevaluate paracellular fluxes and extracellular trapping ofradioactivity. For inhibition studies, Tyr, 3-iodo-␣-methylL-tyrosine (IMT), BCH, AIB, MeAIB, or Arg was added ata final concentration of 1 mmol/L. The cells were incubated
for 90 minutes at 37°C with 18.5 kBq 4-[125I]mTyr. For the
pH dependence experiment, the pH (hypothetic urine pH) ofapical side incubation medium was adjusted to 5.0, 6.0, 7.0,
or 8.0 by adding HCl or NaOH solution. The pH of thebasolateral side was fixed at 7.4 (hypothetic blood pH). The
cells were incubated for 90 minutes at 37°C with 18.5 kBq
4-[125I]mTyr added to apical or basolateral side.
For transport measurements, an aliquot (50 ␮L) of the
incubation medium on the other side was taken at the
specified time, and the radioactivity was counted. For accumulation studies, the medium was removed by suction at
the end of the incubation period, and the monolayers wererapidly washed twice with 2 mL of ice-cold incubation
medium on each side. The cells on the filters were solubilized in 0.5 mL of 1 N NaOH, and the radioactivity of eachaliquot (100 ␮L) was counted. The 125I and 14C radioactivity of the collected media and the solubilized cell monolayers was determined using an ARC-1000M well-type scintillation counter (Aloka, Tokyo, Japan), and an LS6500
liquid scintillation counter (Beckman Instruments, Fullerton, CA) in Clear-sol II (Nacalai Tesque Inc., Kyoto, Japan).
Inhibition experiments were also performed using natural amino acids as inhibitors. LLC-PK1 cells were seeded on
60-mm diameter plastic tissue culture dishes at a cell density of 5 ⫻ 105 cells/dish in 5 mL of complete medium, andwere used on days 6 and 7 after inoculation. After removalof culture medium, each dish was washed once with 5 mLof incubation medium for 10 minutes at 37°C. Uptake wasmeasured in the 2 mL of Na⫹-free incubation medium. Oneof the natural amino acids was added to a final concentrationof 1.0 mmol/L. Cells were incubated for 10 minutes at 37°C
with 18.5 kBq of 4-[125I]mTyr. Thereafter, media wereaspirated, and monolayers were rapidly rinsed twice using 5
mL of ice-cold incubation medium. Cells were solubilized
in 1.5 mL of 1 N NaOH, and the radioactivity of eachaliquot was counted. Radioactivity associated with solubilized cells was determined using an ARC-1000M well-type
scintillation counter (Aloka).

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Fig. 3. Growth curve of LLC-PK1.

3. Results
The labeling efficiency of 4-[125I]mTyr was greater than80%. After purification, no-carrier-added 4-[125I]mTyr withradiochemical purities greater than 95% was obtained, andspecific radioactivity was greater than 8.1 ⫻ 1019 Bq/mol.
Figure 3 shows the growth curve of LLC-PK1. It took 4days to reach confluence. Dome structure attributed to reabsorption of water appeared 4 days after inoculation (Fig. 4).
Figure 5A shows the transcellular transport of 4-[125I]mTyr from the basolateral to apical side, and from the apicalto basolateral side. Apical-to-basolateral transport of4-[125I]mTyr (26.66 ⫾ 2.82%) was much greater than basolateral-to-apical transport (11.65 ⫾ 0.54%) for up to 120
minutes. Incubation at 4°C significantly decreased transcellular transport and accumulation of 4-[125I]mTyr (P ⬍0.001). Accumulation of 4-[125I]mTyr from the basolateralside (1.30 ⫾ 0.08%) and apical side (2.19 ⫾ 0.02%) was
observed at 90 minutes of incubation (Fig. 5B). Apical-to-

2.6. Statistical analysis
The values obtained in each experiment were expressedas the mean ⫾ SD. Statistical comparisons between groups
were performed using the Student t test.

Fig. 4. Dome structure of LLC-PK1 cell monolayers attributed to reabsorption of water.

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N. Shikano et al. / Nuclear Medicine and Biology 31 (2004) 477– 482

Fig. 6. Effects of inhibitors (1 mmol/L) on transcellular transport (A) andaccumulation (B) of 4-[125I]mTyr. Each column represents mean ⫾ SD of
3-5 LLC-PK1 cell monolayers grown on a permeable support. *P ⬍ 0.01.

mulation of 4-[125I]mTyr from the basolateral or apical sideof the monolayers.
As shown in Fig. 6, at 1 mmol/L, the amino acids BCH,
Tyr, and AIB inhibited accumulation (P ⬍ 0.01) as well as
both basolateral-to-apical and apical-to-basolateral transport(P⬍ 0.01) of 4-[125I]mTyr. IMT also inhibited 4-[125I]mTyrtransport and accumulation from both sides (P ⬍ 0.01).
MeAIB did not exhibit a significant inhibitory effect. Na⫹dependent transport did not significantly contribute to thebidirectional transport.
We evaluated effects of apical-side pH on transcellulartransport and accumulation of 4-[125I]mTyr. As shown in
Fig. 7, when pH of apical incubation buffer was varied from
5.0 to 8.0 (pH of basolateral side fixed at 7.4), accumulationof 4-[125I]mTyr from both basolateral and apical side wassignificantly affected (P ⬍ 0.001), whereas basolateral-toapical and apical-to-basolateral transport were not significantly affected.
As shown in Fig. 8, uptake of 4-[125I]mTyr decreased(⬍30%) in the presence of neutral L-amino acids with large,

Fig. 5. Transcellular transport of 4-[125I]mTyr (A) and accumulation at 90
minutes of incubation (B), paracellular fluxes (C) and the extracellular
trapping of [14C]inulin (D). Values represent the mean ⫾ SD (n ⫽ 4-5
monolayers). *P ⬍ 0.001.

basolateral and basolateral-to-apical leak of [14C]inulin was⬍ 0.26% and ⬍ 0.07% of the total [14C]inulin, respectively,
up to 120 minutes (Fig. 5C). The amount of [14C]inulin
trapped in the extracellular space from both sides was ⬍0.003% of the total [14C]inulin (Fig. 5D).
To examine substrate specificity of the basolateral andapical amino acid transport system in LLC-PK1 cells, weevaluated effects of several amino acids (added to the basolateral or apical side) on transcellular transport and accu-

Fig. 7. Transcellular transport (A) and accumulation (B) of 4-[125I]mTyrdepending on apical pH. *P ⬍ 0.001.

N. Shikano et al. / Nuclear Medicine and Biology 31 (2004) 477– 482

Fig. 8. The effects of natural L-amino acids (1 mmol/L) on 4-[125I]mTyrtransport in apical membrane of LLC-PK1 cell monolayers on dishes.

branched, or aromatic neutral side chains (i.e., Cys, Leu, Ile,Phe, Met, Tyr, His, Trp, and Val).

4. Discussion
As shown in Fig. 5, apparent bidirectional transcellulartransport and accumulation of 4-[125I]mTyr were observedin the LLC-PK1 cell monolayers. Lowered temperature significantly inhibited transport and accumulation, indicatingthat transport of 4-[125I]mTyr is due to carrier-mediatedtransport. Little of the administered dose of [14C]inulin
leaked to the opposite side of the LLC-PK1monolayer. We
attribute this and appearance of a dome structure on dishes
4 days after inoculation to formation of tight junctions in
LLC-PK1monolayers (Fig. 4).
In intact epithelial cells in the proximal tubule, basolateral membrane and apical membrane have different roles.
Basolateral membrane is involved in transport between
blood and tissue, and apical membrane is involved in transport between body and external environment. It is believedthat these functions are related to the finding that basolateral
membrane transport involves the L, A, and ASC systems,
whereas in the apical membrane the b0⫹, B0⫹, B0, A, andASC systems contribute to neutral amino acid transport. In
apical membrane of proximal tubule cells, these transportsystems are predominantly involved in reabsorption of neutral and basic amino acids, including aromatic amino acids.
System b0⫹ is a Na⫹-independent transport system involvedin transport of neutral amino acids (Leu, Ile, Val, Phe, Tyr,
Trp, His, Met, Gln, Asn Thr, Cys, Ser, and Ala) and basic
amino acids (Arg, Lys and Orn) [10]. System B0 is a neutralamino acid transport system predominantly involved in reabsorption of neutral amino acids on the apical side ofproximal tubule cells [11,12]. Palacinet al have suggested
the presence of a Na⫹-dependent transport system (System
B0,⫹) on the apical side of proximal tubule cells [13].
Mammalian L-type amino acid transporter 1 (LAT1) andL-type amino acid transporter 2 (LAT2), which are components of system L, are typical transporters for their respective roles. LAT1 is a major route for providing cells with

481

large neutral amino acids [14,15]. The finding that LAT1 isconspicuously up-regulated in many kinds of tumor andtransformed cell line is consistent with the observed increase in uptake of amino acids for rapid cell growth andproliferation [14]. LAT2 is responsible for neutral aminoacid transport in basolateral membrane of proximal tubule
cells, and for transferring smaller amino acids such as Ala,
Ser, and Thr as well as large, branched, or aromatic neutralamino acids [15]. LAT1 and LAT2 require heavy chain of
4F2 cell surface antigen (4F2hc), a chaperone-like protein,for functional expression [14,15].
Uptake of [14C]Leu via LAT1 is inhibited by Cys, Leu,
Ile, Phe, Met, Tyr, His, Trp, and Val. Ala, Ser, and Thr have
weaker inhibitory effects on LAT1-mediated [14C]Leu uptake [14]. In contrast, Ala, Ser, Thr, Cys, Leu, Ile, Phe, Met,
Tyr, His, Trp, Val, Asn, and Gln strongly inhibit LAT2mediated [14C]Leu uptake [15]. Data reported by Sepulveda
et al. show that Ala and Ser weakly inhibit Leu uptake [5],suggesting that Na⫹-independent uptake of neutral aminoacids by LLC-PK1 cells preferentially targets large andbranched neutral amino acids as substrates (LAT1 substrates rather than LAT2 substrates). In the present inhibition experiment, transport of 4-[125I]mTyr was inhibited by
BCH, L-Tyr and AIB, which are substrates of system L(Fig. 6). Furthermore, uptake of 4-[125I]mTyr decreased in
the presence of large, branched, or aromatic neutral aminoacids (Fig. 8). This substrate selectivity is very similar toreported selectivity of human and rat LAT1 [14,16]. LAT2
with 4F2hc is the prominent system L transporter in thebasolateral membrane of intact proximal cells. However, thepresent results do not clarify the functional expression of
LAT2, because although 4-[125I]mTyr clearly has an affinity
to LAT1, it is unclear whether 4-[125I]mTyr has an affinity
to LAT2. On the other hand, the similarity in inhibition
between bidirectional transport and accumulation of
4-[125I]mTyr (Fig. 6) and the inhibition profiles of naturalamino acids (Fig. 8) suggest that LAT1 with 4F2hc ispresent on both sides of LLC-PK1 cell monolayers. This
arrangement differs from that of intact epithelial cells in
proximal tubule and is consistent with a previous report ofsystem L-mediated transport of L-dopa, a neutral aminoacid, in LLC-PK1 cells [17]. It also indicates that the same
carrier transports 4-[125I]mTyr, L-Tyr, and IMT (Fig. 6).
Lowered pH enhances uptake of Leu via system L [18].
As shown in Fig. 7, transcellular transport and accumulationfrom both directions were enhanced by lowered pH (P ⬍0.001). These observations show that transport and accumulation of 4-[125I]mTyr across the apical membrane can be
stimulated by acidifying the medium on the apical side; that
is, an inwardly directed H⫹ gradient acts as a driving force
for extrusion. This property also supports very high expression of system L–like transporter in LLC-PK1 cells.
Transport of neutral amino acids by LLC-PK1 cells is
dominated by Na⫹-independent transport, most likely involving LAT1, as indicated by the inhibitory effect of BCH
under Na⫹-free conditions. Studies of transport systems in482

N. Shikano et al. / Nuclear Medicine and Biology 31 (2004) 477– 482

the apical membrane of LLC-PK1 cells have shown thepresence of system A and ASC [3,19], and the absence ofNa⫹-dependent neutral amino acid transport systems (B0⫹
and/or B0) of the type responsible for Leu reabsorption by
the proximal tubule in vivo [3,4,19]. In the present study, weobserved very minor uptake (not significant) of 4-[125I]mTyr into LLC-PK1 cells via Na⫹-dependent transport systems (Fig. 6). A lack of affinity of 4-[125I]mTyr for system
A is suggested by the insignificant inhibitory effect of
MeAIB (Fig. 6). AIB, a substrate of system ASC, had asignificant inhibitory effect (Fig. 6). The results shown in
Fig. 6 indicate that 4-[125I]mTyr transport and accumulation
via Na⫹-independent transport systems are inhibited by
AIB. Expression of system ASC could not be excluded[3,4,19], but all transport of 4-[125I]mTyr was Na⫹-independent. This suggests that 4-[125I]mTyr does not have anaffinity with system ASC.
Several cell lines have been proposed as alternatives to
LLC-PK1, including MDCK and OK [5]. Characteristics ofkidney epithelial cell lines depend on the renal tissue fromwhich they are derived. These cell lines usually have different characteristics from intact cells of those tissues (e.g.,
up-regulation and defects of transporters), and these differences may affect results. With appropriate cell lines, thepresent experimental system is useful for studying transportof radiopharmaceuticals.
In conclusion, we investigated transcellular transport of4-[125I]mTyr in LLC-PK1 (porcine kidney epithelial cellline) monolayers grown on collagen-coated microporous
membrane filters. We observed bidirectional transcellulartransport of 4-[125I]mTyr by LLC-PK1 cell monolayers. The
system that transported 4-[125I]mTyr also transported L-Tyr
and IMT. Na⫹-dependent 4-[125I]mTyr uptake was insignificant. Transport of 4-[125I]mTyr by LLC-PK1 cells is dominated by system L–like Na⫹-independent transport (mostlikely via a porcine homologue of LAT1).
Acknowledgments
The authors thank Yuri Aisawa, Toshio Miyamoto,
Takuya Doi, and Daisuke Izumi (Ibaraki Prefectural University) for their excellent technical assistance. This work
was supported by Grants-in-Aid for Scientific Research(10770451, 14770498, 13557075, and 15659283) from the
Ministry of Education, Science, Sports, and Culture of Japan and the Japan Society for the Promotion of Science.
Financial support was also provided by Ibaraki Prefectural
University Project Research (9808-3, 0118-1, and 0220-1)
and Ibaraki Prefectural University Grants-in-Aid of the Encouragement for Young Scientists 2001 and 2002.

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